Studies on drug resistance in HIV-related Kaposi sarcoma

卡波西氏肉瘤(KS)是一种由人类疱疹病毒8型引起的肿瘤，通常发生在免疫系统减弱的艾滋病患者中。在艾滋病相关的KS中，高效抗逆转录病毒疗法(HAART)可以显著地诱导疾病的消退；然而，HAART并不总是有效的，KS的二线治疗包括化疗干预。多柔比星等蒽环类药物经常用于艾滋病相关的KS的治疗，但确实会出现对这些药物的耐药性。紫杉醇和多西紫杉醇等紫杉烷类药物已成功用于治疗阿霉素耐药的KS。然而，还没有研究探讨KS对紫杉烷类药物的潜在耐药机制。由于在治疗艾滋病相关的KS时，HAART经常与化疗同时使用，因此检查最近批准的抗病毒药物对用于治疗KS的化疗的潜在耐药机制的影响是重要的。我们对Kaposi肉瘤耐药机制的兴趣来自于我们之前对ATP依赖的转运体P-糖蛋白(P-gp)的研究。我们以前证明了P-gp可以阻碍HIV病毒的产生，并降低过度表达转运蛋白的细胞的传染性。此外，一些HIV蛋白酶抑制剂已被证明是P-gp的底物。在卡波西肉瘤的一个细胞系模型中，我们发现蒽环类药物治疗会导致P-gp表达增加，而HIV蛋白酶抑制剂可以进一步增强这种表达。然而，用于这项研究的细胞系最近被证明是错误的，因此需要重新评估这些结果。我们目前的工作集中在L1T2细胞系上，该细胞系已被证明在小鼠模型中概括了卡波西肉瘤的临床特征。我们用Taq-MAN低密度阵列微流控芯片检测了380个耐药相关基因的mRNA表达，包括参与药物外排、DNA修复机制、细胞凋亡和代谢酶等基因的表达，从而确定了L1T2细胞株的耐药亚系。FACS分析和Western Blot分析显示，在紫杉醇和奈非那韦(蛋白酶抑制剂)选择的细胞中，ABCB1(P-糖蛋白)的表达增加。药物转运蛋白活性分析显示，在用奈非那韦或紫杉醇选择的细胞中，由于P-gp转运蛋白活性较高，罗丹明123的外流较高。根据这些结果，长期使用奈非那韦筛选的细胞对紫杉醇表现出更高的耐药性，而加入P-gp抑制剂valspadar使这些细胞对药物更敏感。这些结果提示，HIV蛋白水解酶抑制剂可能在诱导化疗耐药中起作用。最终，我们希望识别卡波西肉瘤的新耐药机制将导致改进的治疗方法。

Kaposi's Sarcoma (KS) is a type of tumor caused by the human herpesvirus 8 that often develops in AIDS patients with weakened immune systems. In the case of AIDS-related KS, highly active antiretroviral therapy (HAART) can induce significant regression of the disease; however, HAART is not always effective and second-line therapies for KS include chemotherapeutic intervention. Anthracyclines such as doxorubicin are frequently used in the treatment of AIDS-related KS but resistance to these agents does arise. Taxanes such as paclitaxel and docetaxel have successfully been used to treat doxorubicin-resistant KS. However, no studies have examined the potential resistance mechanisms to taxanes in KS. As HAART is often administered concomitantly with chemotherapy when treating AIDS-related KS, it is important to examine the effects of recently-approved antivirals on potential resistance mechanisms to chemotherapy used to treat KS. Our interest in resistance mechanisms in Kaposi's sarcoma comes from our previous work with the ATP-dependent transporter P-glycoprotein (P-gp). We previously demonstrated that P-gp can hamper HIV virus production and decrease infectivity in cells that overexpress the transporter. Additionally, a number of HIV protease inhibitors have been shown to be substrates of P-gp. In one cell line model of Kaposi's sarcoma, we found that anthracycline treatment leads to increased P-gp expression and that HIV protease inhibitors can further augment this expression. However, the cell line used for this study was recently shown to be mischaracterized, thus requiring a reevaluation of these results. Our current work focuses on the L1T2 cell line that has been shown to recapitulate clinical features of Kaposi's sarcoma in mouse models. We have characterized the drug resistance pattern of this cell line selected for resistance to paclitaxel and HIV protease inhibitors and have isolated resistant sublines of the L1T2 line to identify potential mechanisms of resistance using a Taq-man low-density array microfluidic chip to detect mRNA expression of 380 drug resistance-related genes comprising genes involved in drug efflux, DNA repair mechanisms, apoptosis and metabolizing enzymes. FACS analysis as well as Western Blot analysis show increased expression of ABCB1 (P-glycoprotein) in both paclitaxel and nelfinavir (protease inhibitor)-selected cells. Drug transporter activity assays show higher efflux of Rhodamine123 in cells selected with nelfinavir or taxol due to higher P-gp transporter activity. In accordance with those results, cells under prolonged selection with Nelfinavir showed higher resistance to taxol, whereas addition of valspadar, a P-gp inhibitor, made these cells more sensitive to the drug. These results suggest that HIV protease inhibitors may play a role in inducing resistance to chemotherapy. Ultimately we hope that identification of novel mechanisms of resistance in Kaposi's sarcoma will lead to improved therapies.